A Combined biochemical and genetic approach is planned to characterize mutations in the gene for hypoxanthine phosphoribosyltransferase (HPRT, E.C.2.4.2.8) in human cells. We have previously described methods to identify, isolate, and characterize mutant forms of HPRT protein in human cells. We are now concentrating on developing methods to examine the DNA sequence in normal and mutant forms of the HPRT gene. Specifically, we have three major projects underway: A. The Purification of Human HPRT mRNA: We are using sucrose gradient centrifugation and agarose gel electrophoresis to purify HPRT mRNA. The HPRT mRNA is detected by in vitro protein synthesis in a rabbit reticulocyte lysate system, separation of the labeled HPRT from the majority of the other proteins by Sepharose conjugated with anti-HPRT antibody, followed by SDS polyacrylamide gel electrophoresis and autoradiography. B. The Synthesis of a cDNA Copy of Human mRNA: We are synthesizing cDNA copies of the enriched mRNA species obtained in Section A above, and we plan to clone the cDNA species in a bacterial plasmid and isolate the one corresponding to HPRT. C. Developing a Defective Polyoma Virus to Clone the Human HPRT Gene: We have prepared a defective polyoma vector by deleting the "late" region of the viral genome.